In this request for renewal of MH 25998, we plan to expand our investigation of the regulation of the catecholamine biosynthetic enzymes. Our work will proceed as follows: 1. Genetic regulation. We will study the genetic controls on steady-state enzyme levels in inbred mouse strains. We have established that tyrosine hydroxylase (TH), dopamine Beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) are each under the control of a single gene which may regulate the level of all three enzymes in concert. Genetic control of PNMT levels is determined by a gene which regulates the rate of degradation of this enzyme. DBH levels are also genetically determined; biochemically this control may also be expressed on the degradation of the enzyme. The reaction mechanism is more complex than that of PNMT, however, and may proceed by a two-step inactivation process. 2. and 3. Regulation of synthesis and degradation of DBH and PNMT. We have shown that both DBH and PNMT are regulated by two mechanisms: (a) transsynpatic induction of enzyme synthesis, a mechanism which permits elevation of the enzymes above steady-state levels and (b) hormonal control by adrenal cortical glucocorticoids, which regulate steady-state enzyme levels by inhibiting in vivo DBH and PNMT proteolysis. Each of these regulators is expressed through a cholinergic receptor on the adrenal cortical and adrenal medullary cells. We plan to characterize these receptors and to isolate the protease(s) involved in degradation of DBH and PNMT. In addition we have obtained evidence that glucocorticoid control of PNMT degradation is expressed through a stabilizing factor which is lost after hypophysectomy. This factor appears to bind to PNMT, protecting the enzyme against in vivo proteolysis. We have also partially isolated a protease which degrades purified PNMT and may be the enzyme responsible for its in vivo degradation. We will continue our efforts to isolate this enzyme as well.